I was born in the lovely coastal city of Charleston, S.C. in 1916 and lived there until I was thirteen. In Charleston I first became enamored of “natural history” when I attended nature study classes and field trips to nearby beaches, marshes and woods, sponsored by the Charleston Museum. I became an avid shell collector and bird watcher (that was before the term “birder” was coined), and I still enjoy these hobbies. In 1929, my family moved from Charleston to Orangeburg, S.C., an inland, rural town of about 8,000 inhabitants, where my mother had grown up and still had some family. The reason for the move was that the Furchgott department store in Charleston, which had been started by my grandfather and was being run by my father and his two brothers, was unable to survive in the midst of the Depression, and my father decided to open a women’s clothing store in Orangeburg. So I spent my high school years in Orangeburg, enjoying small town life and competing with my first cousin Edwin Moseley for the highest grades in our class. He won.
Within the first couple of years of high school, I knew that I would like to be a scientist. My parents were encouraging: they gave me chemistry sets and a small microscope as presents. I liked to read popular books about scientists, although there were not many available at that time. My father subscribed to the Sunday New York Times, in which there was often a column on science that I found very exciting.
During the four years that I was in high school, my older brother Arthur was at the University of North Carolina at Chapel Hill. I wanted to attend college there also, but that was not possible when I finished high school in 1933 because tuition for me, as an out-of-state resident, was more than my father could afford at that time. So I spent my freshman year at the University of South Carolina, where my tuition was much less. However, by the summer of 1934, my father moved his business from Orangeburg to Goldsboro, N.C., where he felt that the local economy was better. So now, as a resident of North Carolina, I was able to register at the University at Chapel Hill as a sophomore majoring in chemistry.
At Chapel Hill, I had a number of excellent teachers in chemistry. During my junior and senior years, I had a small amount of financial support from an NYA job (NYA being the initials of the National Youth Administration set up by the federal government to help students during the Depression). In that job, I was a lab assistant in research to a junior faculty member working on the physical chemistry of solutions of cellulose. I had decided early in my college years that I would go on to graduate work in some branch of chemistry. My preference by the time I was a senior was physical organic chemistry. I sent letters to dozens of chemistry departments applying for a graduate fellowship or teaching assistantship. I had an excellent academic record, but by graduation time I still had no definite offer of a position for graduate training. I was almost resigned to taking a job in chemical industry, when around the middle of June while I was in Florence, S.C., where my parents now lived, an unexpected offer of a teaching assistantship came to me from the Physiological Chemistry Department of Northwestern University Medical School in Chicago. I was to be a graduate student of Dr Henry Bull, who had recently come to Northwestern, and whose research interests were physical chemical aspects of biochemistry.
Northwestern and Cold Spring Harbor (1937-1940)
Before I went to Chicago, I worked for two summer months in 1937 for Eastern Airlines at the Philadelphia airport – a job which my older brother Arthur, who was employed by that airline, helped me obtain. The job allowed me to save some money and also allowed me free air travel to Chicago. That helped a lot since my stipend as a teaching assistant at Northwestern was only $50 a month for a nine-month academic year. When I arrived in Chicago, it had already been arranged for me to share a room with two more advanced graduate students. Living in Chicago was quite a change from living in the Carolinas. When I would walk to work in the winter from our rooming house, which was about a mile from the medical school, the chill wind whipping in from Lake Michigan along Chicago Avenue was quite an experience for a Southern boy.
My course work at Northwestern was partly at the medical school, and partly at the Evanston campus to which I would travel via the El. At the Evanston campus, my courses were mainly in physical chemistry under Dr Malcolm Dole, who was also on my PhD advisory committee. At the Chicago campus, I had to take physiology and bacteriology (along with medical students), Henry Bull’s course on physical chemistry in biochemistry, and some assorted graduate courses in physiology and biochemistry. The physiology course was under the direction of Dr Andrew Ivy, who had built up a sizeable physiology department faculty for those times. In contrast, the biochemistry faculty consisted only of the chairman, Dr Chester Farmer, Dr Bull and two part-time lecturers.
My laboratory work with Bull started out with the preparation of purified egg albumin. He was studying physical chemical changes in this protein after different methods of denaturation. He had begun to involve me in some of his studies when the summer of 1938 came along, and that turned out to be a special summer for me. Bull had been invited to present a paper on his work at the sixth Cold Spring Harbor Symposium on Quantitative Biology which was to take place at the Cold Spring Harbor Biological Laboratory of the Long Island Biological Association. The theme of the symposium, which was to run for five weeks in a leisurely fashion was the structure and function of proteins. Bull had obtained permission from the director of the Cold Spring Harbor Laboratory, Dr Eric Ponder, for me to attend the symposium, while earning my room and board by running the lantern slide projector at the lectures. The symposium was very exciting. I met many distinguished scientists. Ponder and a physician-scientist, Harold Abramson, arranged to have me assist in a research project at the laboratory for the rest of the summer after the symposium was over. The project was on the electrophoretic mobility of rabbit erythrocytes and ghosts, measured with the use of a microelectrophoresis cell and light- and dark-field microscopy.
By the end of the summer, I had become very interested in the physical chemistry of the red blood cell membrane. When I returned to Northwestern in the fall of 1938, Bull approved continuation of my research on red blood cells as a PhD thesis project. In particular, I was fascinated by the unexplained phenomenon of the transformation of mammalian red blood cells, suspended in unbuffered isotonic saline from discs to perfect spheres when a small drop of the suspension was placed between slide and coverglass. I discovered that the disc-sphere transformation depended on two factors. The first was a rise in pH to over 9.0 in the unbuffered suspension, as a result of the alkaline nature of the glass surfaces (pH being measured with a semi-micro glass electrode that I constructed). The second factor was the removal from the suspension of the red blood cells by adsorption onto the glass surfaces of the slide and coverglass of a substance in the suspension that prevented sphering on elevation of pH of the suspension. I demonstrated that this substance, which I termed the anti-sphering factor, was serum albumin which could not be effectively removed from the red cells simply by multiple washing and centrifuging. In addition to the work on shape changes in erythrocytes, my PhD thesis work involved additional studies on the electrophoresis of the cells under various conditions and on other aspects of the physical chemistry of erythrocyte membranes.
In the summer of 1939 at the invitation of Ponder, with whom I had extensive correspondence during the year and who had become in effect the major advisor for my PhD thesis research, I returned to Cold Spring Harbor to continue research on red blood cells. To earn my room and board, I waited on tables in the communal dining room. I also was able to attend the symposium talks of that year, which were on the subject of biological oxidations. There I first became aware of the new developments in oxidative energy metabolism and the importance of high energy phosphate compounds. Among the many outstanding biochemists attending were L. Michaelis, Fritz Lipmann and Carl Cori. Ponder and his young wife Ruth were very hospitable to me. I was much impressed with his skill in applying mathematics in his research, his facility in scientific writing, and his large collection of records of classical music.
I was able to complete and defend my thesis in time to receive the Ph.D. degree in June of 1940. Earlier that spring I had attended the annual meeting of the Federation of American Societies for Experimental Biology (FASEB) in New Orleans. I had fortunately been asked by Henry Tauber, an Austrian biochemist working for a pharmaceutical firm in Chicago, to share the driving in his car on the round trip to New Orleans as well as his room in a rundown hotel in New Orleans. Thus, I was able to attend this meeting at very little expense. At the FASEB meeting in New Orleans, where gatherings of participants were still called “smokers” and even a fancy meal was not more than two dollars, I had some interviews with persons about possible post-doctoral jobs. One of the interviews was with Dr. Ephraim Shorr, an Associate Professor of Medicine at Cornell University Medical School in New York City, whom I had met at Cold Spring Harbor the summer before. A few weeks later Shorr offered me a postdoctoral position in his laboratory. Although I was hoping to get a position which would allow me to continue work on physical chemistry of proteins or cell membranes, none came through, and I accepted the position with Shorr, with the understanding that I would begin in September.
The reason for waiting until September to begin work at Cornell was because I wanted to spend one more summer at the Biological Laboratory at Cold Spring Harbor. This time, however, I went there as an invited speaker at the symposium which that summer was on the topic of permeability of cell membranes. My talk was entitled “Observations on the structure of red cell ghosts.” At that symposium, there were again a number of established distinguished scientists like K.S. Cole, Robert Chambers and F.O. Schmitt; and in addition, a number of bright young scientists like Hans Neurath, who had also been at the 1938 symposium, Hugh Davson, who with Danielli had developed the lipid bilayer membrane model, and Benjamin Zweifach, with whom I was to collaborate later in research.
Cornell University Medical College (1940-1949)
I stayed at Cornell University Medical College working in the laboratory of’ Ephraim Shorr for nine years. When I arrived, Sam Barker, a young research associate, was there to instruct me in methods and procedures they were using to study tissue metabolism (largely using Warburg manometers) and the turnover of rather ill-defined tissue organic phosphate fractions from canine cardiac muscle during incubations in vitro. For such studies the lab was one of the first to use radioactive phosphate, which we obtained from the cyclotron laboratory at Berkeley. Barker left toward the end of my first year at Cornell; and I was then responsible for running the laboratory for Shorr. Shorr himself, would sometimes take part in preparing tissue for the Warburg experiments. He was quite capable in the laboratory in addition to being a busy and excellent clinician.
During my first two years at Cornell, my major project was on phosphate exchange and turnover, using radioactive phosphate and slices of dog left ventricular muscle. A full paper on the work was published in the journal of Biological Chemistry in 1943. The methods and equipment we used in that work have long been superseded, but we did manage with chemical and some early enzymatic methods to show the extremely fast turnover of creatinine phosphate and the terminal phosphate of ATP in resting cardiac muscle.
The 1943 paper was my first full publication after three years of work at Cornell. One likely reason for sparse output was that the United States had entered World War II in December of 1941, and Shorr, like many others, began to undertake research that had more relevance to the war effort. With government and other support, he shifted the major research in the lab to circulatory shock – first on changes in tissue energy metabolism resulting from hypoxia associated with hemorrhagic shock, and then mainly on factors that might account for “irreversible” shock, the condition in which restoration of blood volume is no longer able to raise pressure and sustain life in the animal subjected to maintained low blood pressure as a result of controlled hemorrhage. To help in this new line of research, Shorr recruited Benjamin Zweifach, then a bright young physiologist who had trained with Robert Chambers and had developed a beautiful method for microscopic observation of blood flow in part of the mesentery (the “mesoappendix” area) of the anesthetized rat. In brief, the “rat mesoappendix test”, conducted by Zweifach and technicians whom he trained, produced evidence by 1944 for two vasoactive factors in circulatory shock. The first factor appeared in the plasma of dogs in the early reversible (by transfusion) stage of hemorrhage. Intravenous injections of this plasma increased the sensitivity of the small arterioles and pre-capillary sphincters to topically applied epinephrine in the mesoappendix test. This factor was termed VEM (for vasoexcitatory materials). As the irreversible stage of circulatory shock developed, VEM activity disappeared from the plasma and a new factor appeared which markedly decreased the sensitivity to epinephrine in the mesoappendix test. This factor was termed VDM (for vasodepressor material). We developed evidence, in part from in vitro experiments with tissue slices, that hypoxic kidney was the probable source of VEM and that hypoxic liver was the probable source of VDM. By late 1945, these developments led to a lead article in the journal Science by Shorr, Zweifach and myself.
During the war years, I was not solely involved in research on tissue metabolism and circulatory shock. In 1943, Eugene DuBois, chairman of the Department of Physiology at Cornell, arranged that I join his department as an instructor in order to replace a staff member lost to military service. Although I was teaching in physiology, I still spent most of my time in research in Shorr’s lab, which was partially funded by the federal Office of Scientific Research and Development. The work on VEM and VDM continued after the war ended. I had attempted to isolate the VEM-like material that accumulated in incubation fluid when kidney slices were incubated anaerobically. I was able to concentrate it somewhat and it appeared to be a labile dialyzable peptide, but I failed to isolate it. On the other hand, Abraham Mazur, a professor of biochemistry at the City College of New York who worked part time with us, purified a VDM-like material from liver which appeared to be ferritin. (Ferritin or not, we might now wonder whether VDM could somehow be related to nitric oxide!)
Unfortunately, the only bioassay procedure for detecting VEM and VDM activity was that involving changes in sensitivity to epinephrine in the rat mesoappendix test. Intravenous injections of solutions containing high levels of impure VEM or purified ferritin did not effect blood pressure in experimental animals. Attempts to develop an in vitro bioassay system also failed. These failures tempered my enthusiasm, and I think that of Zweifach, for the significance of VEM and VDM in the regulation of circulation. However, the failed attempts to develop an in vitro bioassay for VEM and VDM were very important for me for they introduced me to the pharmacology of smooth muscle, a subject that has been a major interest of mine ever since.
Two of the isolated smooth muscle preparations that I unsuccessfully tested for bioassay of VEM and VDM were a helically-cut strip of rabbit aorta, which responded with contraction to epinephrine, and a longitudinal segment of rabbit duodenum, which exhibited spontaneous rhythmic contractions that were inhibited by epinephrine and stimulated by acetylcholine. At that time, contractions of such smooth muscle preparations mounted in organ baths were recorded with isotonic levers on kymographs. One day in the course of making tests on segments of rabbit duodenum mounted in oxygenated Krebs solution, I was surprised to see that during the first hours of the experiment, contraction amplitude did not stabilize as usual but declined gradually and markedly even though the rhythmic frequency remained unchanged. I suspected that my technician had forgotten to add glucose to the Krebs solution. Adding glucose now quickly increased contraction amplitude to the normal level. This finding led to a simple procedure for finding out what sugars and fatty acids could be utilized for energy for contraction in the intestinal smooth muscle under aerobic and anaerobic conditions and to analyze the sites of action of metabolic inhibitors.
In the spring of 1949, 1 had two interesting offers at the assistant professorship level – one in physiology at Duke and one in pharmacology at Washington University School of Medicine. I decided on Washington University, partly because the new chairman there, Oliver Lowry, was someone I had known in the Enzyme Club in New York City and partly because I had begun to be very interested in pharmacology as a discipline. This was partly because of the studies I had begun on the effects of drugs and other agents on smooth muscle preparations in vitro, but also in large part because of my close friendship with Walter Riker, who was then a junior member in the Pharmacology Department at Cornell at the beginning of a distinguished career. His enthusiasm for research in pharmacology was contagious.
In the summer of 1949, my family and I drove from New York to St. Louis. My wife, Lenore, a native New Yorker, said she felt like she was going West in a covered wagon. By that time we had two daughters, ages four and one. Later we had a third daughter born in St. Louis. It might be noted here that none of my daughters became scientists. Instead, they all went into art (like my younger brother, Max). It might also be noted here that my wife Lenore died in 1983; and that now I have a new wife, Margaret (Maggie). I have been very fortunate in having wives who encouraged my work, even though it often reduced the time I could give to family matters.
Washington University (1949-1956)
My seven years in the Pharmacology Department at Washington University were enjoyable ones. Oliver (Ollie) Lowry had been appointed chairman of that department a year or so before I came. He was already well recognized for his ingenuous methods involving enzymology , spectrometry and fluorometry in the quantitative analysis of important enzymes, substrates and products in extremely small amounts of tissue. He was very helpful in introducing me to enzymatic-spectroscopic methods (as developed by kalckar) for analysis of ATP, ADP and AMP. As a new chairman, Lowry inherited two faculty members, Helen Graham and Edward Hunter, and recruited two new ones, namely myself and Morris (Morrie) Friedkin. I had never had a course in pharmacology as a student, much less taught in one, and so I had to spend a lot of time during my first year in St. Louis keeping ahead of the medical students. Later, when I set up my own department in Brooklyn, I adopted for the pharmacology course there much of the lecture, laboratory and conference program that I had participated in at St. Louis.
Lowry’s department was a stimulating place for research. Over the years I was there, the departmental staff grew steadily. Lowry attracted outstanding postdoctoral fellows, such as Eli Robbins and Jack Strominger. We often joined the members of Carl Cori’s Biochemistry Department for seminars and journal club meetings.
My first research project at Washington University was a continuation of the work I had begun at Cornell on energy-metabolism and function of rabbit intestinal smooth muscle. I was able to obtain a small grant to support my research on smooth muscle, and to hire a technician, Marilyn (Wales) McCaman, who later became my first graduate student. By the middle of 1951, my favorite in vitro smooth muscle preparation had shifted from the rabbit duodenum to the rabbit thoracic aorta. I had found that the helical (spiral) strip of that vessel, properly cut and mounted in organ chambers for isotonic recording, gave very reproducible contractions to epinephrine and norepinephrine after equilibration in oxygenated Krebs bicarbonate solution. I had at first planned to study the effects of disturbances in energy-metabolism on these contractions, but I became much more interested in using the aortic strip for studies on drug-receptor interactions.
By 1953, I had published a paper entitled “Reactions of strips of rabbit aorta to epinephrine, isoproterenol, sodium nitrite and other drugs”. Among the other drugs was acetylcholine. I found that it only produced contractions, whether it was added to resting strips or strips precontracted with some other agent. That was a paradoxical response since acetylcholine was known to be a very potent vasodilator in vivo. Little did I suspect then what I was able to show many years later – namely, that relaxation of arteries by acetylcholine is strictly endothelium-dependent, and that my method of preparing the strips inadvertently resulted in the mechanical removal of all the endothelial cells.
In 1954, I published a paper on the use of dibenamine in differentiating receptors in the aortic strip, and in 1955 a review in Pharmacological Reviews on the pharmacology of vascular smooth muscle. In that review, I tried to develop receptor theory as a logical base for interpreting the responses of vascular smooth muscle to many neuro transmitters, hormones and drugs. In order to derive equations to account for the very slow onset and offset kinetics of competitive antagonists as compared to the fast kinetics of agonists, I developed a biophase model in which the agents moved between an aqueous extracellular phase and a lipid membrane phase containing the receptors. Although I paid homage in my review to A. J. Clark for his pioneering work in developing receptor theory, I took issue with his hypothesis that response of a tissue to an agonist is proportional to the fraction of receptors occupied by the agonist. Our results with dibenamine, which behaved as an irreversible competitive blocking agent of adrenergic -receptors, had indicated that with a strong agonist like epinephrine, one could still achieve well over half of the maximum contraction when only a small fraction of receptors were still active. This was the beginning of my interest in the concept of “receptor reserve” or “spare receptors.” (A year later, R.P. Stephenson published his classic paper on the subject in which he introduced the concepts of efficacy, full agonist and partial agonist.)
In the review of 1955, I also briefly reported on a newly discovered phenomenon – namely, that strips of rabbit aorta undergo reversible relaxation when exposed to light of proper wavelength and intensity. This photorelaxation was an accidental discovery that came from the observation that in one experiment active contractile tone of two strips in one pair of organ chambers fluctuated with time, whereas that of two strips in another pair of chambers remained steady. The two strips showing fluctuations did so synchronously. Those two strips, but not the other two, were in organ chambers near a window through which they were exposed to skylight. Suspecting that the fluctuations in tone were due to fluctuations in light intensity on the strips near the window (it was a cloudy-bright day), I closed the shade on the window and both strips increased in tone. I opened the shade and both decreased in tone. From that point on, we never allowed our strips to be exposed to direct skylight. (The usual overhead fluorescent lights do not produce photorelaxation.) Some studies on the characteristics of photorelaxation were begun in St. Louis, and then extended when I moved to Brooklyn.
In addition to working on in vitro smooth muscle preparations at Washington University, I also began what became many years of research on the pharmacology of an in vitro cardiac muscle preparation – namely the isolated electrically-driven right atrium of the guinea pig. In starting that work, I had the assistance of a very able technician, Taisija De Gubareff. Using chemical and enzymatic methods for analysis of creatinine phosphate, ATP, ADP, and AMP, we showed that neither development of “experimental failure” in vitro (a steady loss of contractile force over hours) nor recovery from failure on addition of a cardiac glycoside was due to changes in concentration of these high-energy phosphates. We also reported on the effects of anaerobiosis and of a number of positive and negative inotropic agents. We collaborated with my good friend William Sleator of the Physiology Department in the study of changes in cellular action potentials (measured with intracellular microelectrodes) associated with the changes in contractility of the guinea pig atrium in response to epinephrine and acetylcholine, and a number of other inotropic agents.
Suny Medical Center in Brooklyn (1956-)
In 1956, I accepted the position of chairman of the new Department of Pharmacology at the State University of New York (SUNY) College of Medicine at New York City (actually in Brooklyn, and later changed in name to SUNY Downstate Medical Center and more recently to SUNY Health Science Center at Brooklyn). The department had previously been part of a joint physiology and pharmacology department headed by Chandler Brooks but with the opening of a new, relatively huge (for the time) basic science building for the medical school and with good financial support from the State University, there was ample room and resources for a separate department. From the former joint department, I inherited Julius Belford as an associate professor and Bernard Mirkin as an assistant professor. For additional faculty, I recruited Kwang Soo Lee, Leonard Procita, Lowell Greenbaum, Walter Wosilait and Arthur Zimmerman, all in time for them to teach our first course for medical students. The following year C. Y. Kao joined the staff. Also during the first year, we accepted our first graduate students, namely Maurice Feinstein, who worked with me, and Arnold Schwartz, who worked with Lee. During that year I didn’t do much bench work in the research lab since most of my time was spent organizing the department and learning how to be a chairman. (I never became a well-organized administrator and was always poor at delegating authority.)
In Brooklyn, I continued research on photorelaxation of blood vessels, factors influencing contractility of cardiac muscle, peripheral adrenergic mechanisms, and receptor theory and mechanisms. Then, about twenty-three years after moving to Brooklyn, the research in my laboratory largely shifted to endothelium-dependent relaxation of blood vessels. For convenience, I shall divide the discussion of research in Brooklyn into subsections corresponding to the areas that I have listed.
Photorelaxation of Blood Vessels
Helping with this research were Eugene Greenblatt, my first postdoctoral fellow, and Stuart Ehrreich, my third graduate student. Among other things, we were able to obtain an accurate action spectrum (with a peak at 310 nm) for the photorelaxation. Later we observed that addition of sodium nitrite to the bathing medium greatly sensitized the rabbit aortic strip to photorelaxation and shifted the peak of the action spectrum to about 355 nm. Ehrreich and I found that many other smooth muscle preparations (from stomach, intestine and uterus) which did not ordinarily relax in response to radiation did so in the presence of inorganic nitrite. Percy Lindgren, a visiting faculty member from the Karolinska Institute, also worked with us for a while on photosensitization by nitrite.
Many years later in the early 1980’s, after the discovery of endothelium-derived relaxing factor (EDRF), I again began research on photorelaxation. Although photorelaxation did not depend on the presence of endothelium on the strip or ring of rabbit aorta, we found many similarities between it and endothelium-dependent relaxation (as produced by acetylcholine or A23187). Not only was photorelaxation, like endothelium-dependent relaxation, causally dependent on the elevation of cyclic GMP as a result of stimulation of guanylate cyclase, but both were inhibited by hemoglobin and by methylene blue. This work was carried out with Desingarao Jothianandan, who has been a most helpful research associate in my lab over the past seventeen years. Then, after EDRF was identified in 1986 as nitric oxide, Kazuki Matsunaga (a postdoctoral fellow) and I reinvestigated the potentiation of photorelaxation by sodium nitrite. Using a cleverly designed perfusion-bioassay type apparatus, Matsunaga clearly demonstrated that the potentiation was due to the photoactivated release of NO from nitrite. It is tempting to hypothesize that light (in the absence of added nitrite) produces relaxation of vascular smooth muscle by photoactivating the release of NO from some endogenous compound in the muscle cell.
Factors Influencing Contractility of Cardiac Muscle
My first graduate student in Brooklyn, Maurice Feinstein, did his Ph.D. thesis research on the effects of experimental congestive heart failure, asphyxia and ouabain on high energy phosphates and creatine content of the guinea pig heart. My second graduate student, Albert Grossman, who began work in 1957, did his thesis research on the effects of frequency of stimulation, extracellular calcium concentration and various drugs on calcium exchange and contractility of the guinea-pig left atrium. Grossman and I published three papers based on his thesis research, which was one of the first attempts to determine the rates of exchange of calcium (using 45Ca) between extracellular fluid and various intracellular “pools” of calcium in cardiac muscle under various conditions affecting contractility. We showed that the positive inotropic effects of norepinephrine and strophanthin-K were correlated with an increase in rate of exchange of calcium in an intracellular pool associated with the contractile process and that the negative inotropic effects of acetylcholine and adenosine were correlated with a decrease in rate of exchange in that pool.
We also continued work with ryanodine, which produced a negative inotropic effect on the guinea-pig atrium and actually changed the force-frequency effect from a positive to negative staircase (mimicking the normal staircase in frog heart). Sleator, De Gubareff and I had shown that the decrease in force with ryanodine (unlike that with acetylcholine or adenosine) was not associated with a decrease in duration of the action potential. The thesis research of Grossman and a few years later that of another graduate student, Peter Wolf, also using 45Ca to measure effects of ryanodine on calcium exchange, led to a hypothetical model that fits fairly well with more recent work of others on the reactions of ryanodine with “receptors” involved with calcium transport in the sarcoplasmic reticulum.
Peripheral Adrenergic Mechanisms
In writing the 1955 review on the “Pharmacology of vascular smooth muscle,” I had become very interested in the mechanisms by which sympathetic postganglionic denervation and certain drugs like cocaine markedly potentiate the response of effector organs to epinephrine and norepinephrine, yet markedly reduce the response to the sympathomimetic tyramine. My second postdoctoral fellow, Sadashiv (Sada) Kirpekar, was assigned to work in this area. He proved to be a gifted investigator, and we published a number of papers together on work carried out between 1959 and 1962. In one paper, with the running page heading of “the cocaine paradox,” we presented evidence that in aortic strips of rabbit and isolated electrically-driven atria from guinea pig and cat, cocaine potentiated responses to norepinephrine and inhibited those to tyramine by blocking one and the same site on adrenergic nerve terminals. Blockade of this site inhibited the neuronal uptake of no repinephrine from the region of the adrenergic receptors, thus potentiating its action; however, blockade of the site also inhibited uptake of tyramine, whose sympathomimetic action depends on release of norepinephrine from neuronal storage sites, thus inhibiting its action. The site, which we called the “transfer site” later became known as the uptake-1 (UI) site. In the same paper we showed that reserpine, which depleted neuronal storage granules of norepinephrine, did not interfere with activity of the uptake site. In addition to Kirpekar, Peter Cervoni came in as a postdoctoral fellow to work on peripheral adrenergic mechanisms. Both he and Kirpekar later became faculty members in the department with Kirpekar staying on and becoming a stellar figure in the field of adrenergic mechanisms before his untimely death in 1983.
In 1960, I was invited to present a paper on some of my studies on receptors for sympathomimetic amines at a CIBA Foundation conference on Adrenergic Mechanisms held at CIBA House in London. It was the occasion for my first trip abroad and was very exciting. Among the many distinguished pharmacologists at the conference were Sir Henry Dale, Sir John Gaddum and J.H. Burn. Burn at that time was pushing his “cholinergic-link” hypothesis for norepinephrine release at adrenergic nerve terminals. I felt strongly that he had misinterpreted the experimental results which had led to the hypothesis and in the discussion sessions I presented our own results with isolated atria which indicated that there were nicotinic cholinergic receptors on adrenergic nerve terminals which when stimulated by nicotine or acetylcholine triggered a transient release of norepinephrine, but which played no role in release of norepinephrine on electrical stimulation of the nerve.
In 1962-63, 1 spent a sabbatical year in the Department of Physiology of the University of Geneva, where Jean Posternak was chairman. Although I did some research and teaching there, I spent most of my time writing papers on research that my colleagues and I had completed during the preceding few years and on a review on receptor mechanisms (see below). I also visited a number of laboratories in Europe where outstanding research on adrenergic mechanisms was in progress. Among these were the laboratories of S. von Euler in Stockholm, E. Muscholl in Mainz and John Gillespie in Glasgow.
Between 1965 and 1970 I was fortunate in having a number of very competent coworkers in research on peripheral adrenergic mechanisms. In addition to Kirpekar, there were Pedro Sanchez-Garcia, (a visiting research associate who later became a leading pharmacologist in his native Spain), Jerome Levin (a postdoctoral fellow) and Arun Wakade (a graduate student who later became a faculty member).
In early 1971, I began my second sabbatical leave, this time at the relatively new medical school of the University of California at San Diego (located in La Jolla). I became a visiting professor in Steve Mayer’s Pharmacology Division of the Department of Medicine. One reason for this choice of a sabbatical site was that I wanted to learn the method for analysis of cyclic AMP that Mayer had developed (this was before the development of radioimmunoassays for cyclic nucleotides). However, I did not do a lot of research at La Jolla, partly because a fair amount of my time that year was devoted to duties as president of the American Society for Pharmacology and Experimental Therapeutics.
On returning from La Jolla to Brooklyn in 1972, I continued research on the role of receptors located on prejunctional terminals (varicosities) of adrenergic nerves. I collaborated with Kirpekar in an attempt to characterize the inhibitory prejunctional -adrenergic receptors on the nerve terminals in cat spleen. At the same time, one of my graduate students, Odd Steinsland, was conducting a very exciting thesis project on cholinergic receptors on prejunctional adrenergic nerve terminals in the isolated, perfused central ear artery of the rabbit. He first pharmacologically characterized with the use of various muscarinic agonists and antagonists the prejunctional receptor through which acetylcholine produces a marked inhibition of norepinephrine release (monitored by both the degree of vasoconstriction and [3H]norepinephrine release). He then went on to study the release of norepinephrine from the adrenergic neurons in the ear artery by cholinergic agonists acting on prejunctional nicotinic receptors. At the same time I was continuing studies, with the assistance of Taruna Wakade, on the pharmacology of cholinergic nicotinic receptors on adrenergic prejunctional terminals in the guinea-pig left atrium.
Receptor Theory and Mechanisms
When I first gave a course on receptor theory and mechanisms to graduate students in 1957-1958, the literature on the subject was relatively sparse: papers by Clark, Gaddum, Schild, Ariëns, Stephenson, Nickerson and myself. I became interested in developing suitable theory (occupation theory) and in vitro procedures for differentiating and characterizing receptors. In particular, I concentrated on receptors for adrenergic and cholinergic agents using as test tissues the rabbit aortic strip, duodenal segment, and stomach fundus muscle, and the guinea-pig electrically driven left atrium and tracheal ring.
In 1963, toward the end of my sabbatical year at the University of Geneva, I completed a review on “Receptor Mechanisms” for Volume 4 of the Annual Review of Pharmacology. In it, I took the opportunity to stress the importance of Stephenson’s ideas on efficacy and spare receptors. In 1965 at a symposium on receptor mechanisms at Chelsea College in London, I presented a paper on the use of -haloalkylamines, as irreversible receptor antagonists, in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes. Using a slightly modified form of Stephenson’s equations and introducing a term, , for intrinsic efficacy, I derived a simple equation that predicted that the slope and ordinate intercept of a double reciprocal plot of equiactive concentrations of an agonist before and after irreversible inactivation of a fraction of its receptors, could permit the determination of both the fraction of receptors still active as well as the dissociation constant (KA) of the agonist-receptor complex. For different agonists acting on the same receptor, one could calculate from the KA values the fractional occupation by each to obtain the same standard response before receptor inactivation, and thus obtain relative efficacies. Using this approach, Paula (Bursztyn) Goldberg (a graduate student) and I compared the dissociation constants and relative efficacies of agonists acting on muscarinic cholinergic receptors of isolated strips of rabbit stomach fundus muscle; and later John Besse (a postdoctoral fellow) and I compared the dissociation constants and relative efficacies of agonists acting on 1-adrenergic receptors of rabbit aorta. In light of what is now known about receptor signalling pathways through G-proteins, it is probably better to admit that the pharmacological procedure which we developed for obtaining agonist-receptor dissociation constants can only give approximate relative values. Nevertheless, the procedure has proven useful in a number of studies.
In 1972, I published a review entitled “The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory”. In it I attempted to formulate the methods and necessary conditions for the classification and differentiation of receptors by pharmacological procedures designed to give accurate dissociation constants of competitive antagonists, acting on a given receptor, and accurate relative potencies and, if possible, dissociation constants of agonists acting on the same receptor. In particular, I attempted to point out pitfalls in such procedures and how to avoid them. For example, I derived theoretical equations to illustrate how removal of the agonist from the region of the receptor by active uptake or enzymatic destruction could markedly alter the slope of a Schild plot for competitive antagonism from the theoretical slope of 1. Later, Aaron Jurkiewicz, a visiting research associate from Sao Paulo, Niede Jurkiewicz and I successfully used these theoretical equations in the analysis of propranolol antagonism to isoproterenol in guinea-pig tracheal strips before and after blockade of removal of the agonist by active uptake.
In 1977, I organized for the annual FASEB meeting a symposium on receptors. By then binding of radioligands (usually 3H-labelled competitive antagonists) had been used for several years for quantifying specific receptors in membranes from homogenized cells and for determining the dissociation constants of competitive antagonists and agonists for those receptors. Most of the papers at the symposium were reports of studies with radioligands (e.g., R. J. Lefkowitz on both -and -adrenergic receptors; P. Seeman on dopamine receptors; S. Snyder and colleagues on serotonin receptors and opiate receptors). My paper at the symposium was partly a discussion of how pharmacological procedures for differentiating and characterizing receptors based on occupation theory were still very useful in conjunction with the exciting new developments in receptor research being made with specific radioligands.
Also, I reviewed work that had been carried out in my laboratory on -adrenergic receptors mediating relaxation of guinea-pig tracheal smooth muscle, and presented results of pharmacological experiments that showed that this smooth muscle did not have exclusively the 2-type of the -adrenergic receptor, as dogma of that time would have it, but had an admixture of the 1-type as well – usually as a small fraction of the total of -receptors, but, depending on the guinea-pig used, sometimes much more.
Endo Thelium-dependent Relaxation
Having obtained pharmacological evidence that guinea-pig tracheal smooth muscle sometimes has a sizeable fraction of the 1-type adrenergic receptor along with the 2-type (see above), I decided that it would be well to reexamine the smooth muscle of rabbit thoracic aorta to see if it also might have varying amounts of the 1-type receptor mixed with the 2-type. However, in the very first experiment designed for this new study in May 1978, an accidental finding as a result of a technician’s error completely changed the course of research in my laboratory. The accidental finding was that on the preparation of rabbit aorta being used in the experiment, the muscarinic agents acetylcholine and carbachol induced relaxation rather than the expected contraction. Why this accidental finding was so exciting, how it led to our discovery of the endothelium-derived relaxing factor (EDRF), and how that factor was eventually identified as nitric oxide will not be discussed here since those matters will be considered in detail in my Nobel Lecture.
In 1982, I resigned from the chairmanship of the Department of Pharmacology at the SUNY Downstate Medical Center, but continued as a professor. In 1989, I retired from my professorship (receiving emeritus status), so that I now was free of teaching duties and committee work related to the medical curriculum but could still continue research in the department. My retirement also now allowed me to spend about three and a half months each winter as an adjunct Professor in the Department of Molecular and Cellular Pharmacology of the University of Miami School of Medicine. Most of my time there I have spent trying to catch up on the writing of manuscripts and on the reading of the burgeoning literature in the field of nitric oxide research – an impossible task these days! During the winter sojourns in Miami, I keep in touch with what is going on in my research laboratory in Brooklyn by means of an occasional visit, but mainly by frequent fax and telephone communications with my one or two coworkers there. I consider myself very fortunate in having this Brooklyn-Miami arrangement. Of course, an additional advantage for my wife Maggie and me is that the arrangement allows us to enjoy the very pleasant winter weather in Miami and some of the outdoor activities that it fosters (golf, for instance, in my case).
From 1982 until the present writing, I have been the recipient of a number of honors and awards for my research. Naturally, I have been very pleased to be the recipient. Yet, in thinking back about what aspects of my research have given me the greatest pleasure, I would not place the honors and awards first. I think that my greatest pleasure has come from each first demonstration in my laboratory that experiments designed to test a new hypothesis developed to explain some earlier, often puzzling or paradoxical finding, have given results consistent with the hypothesis. It is not just the immediate pleasure of obtaining such results but also the anticipated pleasure of discussing the results with others doing research in the same area – obviously an ego supportive aspect.
I still enjoy doing bench work in the laboratory with my co-workers. The research still is rather “old fashion” pharmacological research. I was very lucky to stumble on unexpected results in 1978 that led to the finding of endothelium-dependent relaxation and EDRF, and eventually to NO; for if I had not, I would probably have still concentrated on research on receptor theory and mechanisms, and been left far behind by others in that field who have so brilliantly and successfully developed and used molecular biological and other advanced methodologies in their research.
This autobiography/biography was written at the time of the award and later published in the book series Les Prix Nobel/ Nobel Lectures/The Nobel Prizes. The information is sometimes updated with an addendum submitted by the Laureate.
Robert F. Furchgott died on 19 May, 2009.
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